A. Field of Invention
The present invention provides compositions that are useful in preparing and/or serving as antitoxins against the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax.
B. Related Art
B. anthracis has three major routes by which its spores infect mammalian hosts. One route is through cuts or abrasions of the skin, which leads to the generally benign and self-limiting cutaneous form of the disease. The second is through spore ingestion, which generally results in gastrointestinal anthrax. The third is by inhalation of the spores into the lungs, causing inhalational anthrax.
Inhalational anthrax is the deadliest form of the disease. After it is inhaled, the anthrax spores migrate to and germinate within alveolar macrophages that are then trafficked to lymph nodes where the bacterium is able to grow and gain access to the bloodstream. Once in the bloodstream, a serious systemic form of anthrax develops, rapidly leading to death in a high percentage (up to 80%) of cases (Inglesby et al., 2002). As a result, anthrax is listed by the Centers for Disease Control and Prevention as one of six category A agents considered likely to have the most adverse public health impact if used in a biological attack.
Highly virulent forms of B. anthracis encode two types of virulence factors: a poly-D-glutamic acid capsule that protects the bacterium against phagocytosis, and two known anthrax toxins, lethal toxin (LeTx) and edema toxin (EdTx) (Mock and Fouet, 2001). Both LeTx and EdTx are secreted from the bacterium and believed to be primarily responsible for the major symptoms and death associated with anthrax. The toxins are binary, AB-type toxins, consisting of a shared B-moiety (designated a protective antigen or PA) and either of two A-moieties, designated as lethal factor (LF) for LeTX and edema factor (EF) for EdTx. EF is a calcium/calmodulin-dependent adenylate cyclase that causes edema at the site of infection as well as elevated levels of cAMP, thus interfering with bacterial phagocytosis in neutrophils and contributing to bacterial multiplication in the host. LF is a zinc-dependent protease capable of inducing macrophage lysis as well as the cleaving and inactivation of members of the mitogen activated protein kinase kinase protein family (MAPKK). LeTx is also thought to contribute to anthrax pathogenesis by causing macrophages to release pro-inflammatory cytokines and reactive oxygen intermediates believed to cause death by a shock-like syndrome. The precise cause of death by anthrax, however, remains to be established.
The protective antigen (PA) is initially synthesized as an 83 kDa protein that binds specifically, reversibly, and with a high degree of affinity (Kd ˜1 nM) to a receptor on the surface of the host cells. Once bound, the PA is cleaved into two protein fragments by a member of the furin family of proteases. As illustrated in FIG. 1, the N-terminal PA20 fragment is removed and the C-terminal PA63 fragment remains associated with the receptor. PA63 then spontaneously self-associates with other PA63 fragments at the cell surface to form heptameric ring-shaped oligomers that can each bind up to three molecules of either LF or EF. (Mogridge et al., 2002; Cunningham et al., 2002).
The assembled anthrax toxin complexes are then taken up into cells by receptor-mediated endocytosis and are trafficked to an acidic intracellular compartment. At this location, the PA63 heptamer (the prepore) undergoes a dramatic conformational change under the influence of low pH resulting in the formation of a 14-strand transmembrane β-barrel (Petosa et al., 1997; Benson et al., 1998). Concomitantly, EF and LF are translocated across the membrane to the cytosol, an event that is thought to be mediated by the membrane-associated PA pore.
Recent studies have led to the identification of the Anthrax Toxin Receptor (ATR) as a cellular receptor for PA (Bradley et al., 2001). ATR is a type 1 membrane protein with an extracellular von Willebrand factor type A-domain (or integrin-related I-domain) that binds directly to PA, and has been shown to be from the same cellular gene as TEM8 (Tumor Endothelial Marker 8), a ubiquitously expressed gene known to be specifically upregulated in tumor vasculature. To date, three differently spliced mRNAs derived from this gene have been described and designated as TEM8 splice variants 1, 2 (ATR), and 3. The first two splice variants encode transmembrane receptors with different cytoplasmic tails. Each of these proteins can serve as a receptor for anthrax toxin. The third splice variant encodes a protein believed to be secreted from cells as it lacks any obvious transmembrane or other membrane attachment sequence and appears not to mediate anthrax toxin entry.
Due to the high mortality rate associated with inhalational anthrax, there is much interest in developing effective anthrax vaccines. An anthrax vaccine (AVA) representing a sterile supernatant from an attenuated strain of the bacterium is currently licensed for use in the USA. The major protective component of this vaccine is PA (Friedlander et al., 1999). Although the Institute of Medicine has concluded that this vaccine is as safe as others for human administration, it suffers from several major drawbacks, including an 18-month immunization schedule, the need for annual booster doses, and associated symptoms such as headaches, chills, malaise and muscle aches (Vastag, 2002). It may also be possible for bioterrorists to engineer PA in such a way as to alter its antigenic epitopes without changing its function, thereby rendering this vaccine ineffective.
Antibiotics such as doxycycline, penicillin, and ciprofloxacin can also be effective at preventing death by preventing bacterial multiplication (Inglesby, 2002). However, two major drawbacks are inherent in relying solely upon antibiotic therapy. First, antibiotics are effective only if administered early after infection, presumably at a time when fatal levels of toxin have not yet accumulated in the bloodstream. Second, drugs are likely to be ineffective against any organisms engineered to be antibiotic-resistant.
In response to this latter concern, a new approach has been developed to inhibit bacterial multiplication using the PlyG lysin derived from the γ-bacteriophage of B. anthracis to degrade bacterial cell wall components (Schuch et al., 2002). While this approach holds much promise for dealing with the threat of antibiotic-resistant bacteria, it suffers from the same limitation as the use of antibiotics, namely that it would have to be administered early during the course of infection to be effective.
Another approach is to develop antitoxins that can eliminate and/or inactivate anthrax toxins. Currently, four different types of anthrax antitoxin have been described: PA-specific antibodies; soluble receptor-based antitoxins; polyvalent inhibitors (PV1); and dominant-negative forms of PA (DN-PA).
PA-specific antibodies prevent PA binding to its receptor. Recently, high-affinity single-chain variable PA-specific antibody fragments have also been shown to be effective at protecting rats against lethal toxin challenge (Maynard et al., 2002). Such antibodies could prove useful in treating infections by common strains of B. anthracis; however, they would most likely be ineffective against bacteria engineered to express an antigenically-altered form of PA.
Recent studies have also used phage display to identify a dodecapeptide capable of blocking EF/LF binding to the PA heptameric ring (Mourez et al., 2001). In these studies, the multivalent display of this peptide, by covalent attachment to a polyacrylamide backbone (PVI), dramatically increased its inhibitory activity and effectively prevented death of rats challenged with LeTx. Although encouraging, this system is not well suited for multivalent peptide display in humans as acrylamide is highly toxic.
Recent studies involving DN-PA have also described altered forms of PA which harbor amino acid changes that abrogate translocation of anthrax toxin A-moieties into the cytoplasm, without affecting their ability to bind to cell surface associated PA63. These mutant forms of PA are also capable of blocking EF/LF translocation by wild-type PA when they are co-assembled into heptameric rings at the cell surface (Sellman et al., 2001). Presently, this is the most promising class of anthrax antitoxin, especially when one considers the fact that this reagent may also serve as an effective PA-based vaccine.
Finally, soluble forms of ATR have been recently developed to serve as soluble receptor-based antitoxins. In practice, the soluble ATR acts as a “decoy” to compete for and to prevent PA-receptor binding. Studies have shown that such ATR decoys are able to protect cells in culture from being killed by recombinant anthrax toxin (Bradley et al., 2001) and to protect rats against a lethal toxin challenge. Although ATR decoys should be effective inhibitors of anthrax toxin when administered in vivo, they may cause adverse side-effects because they contain the presumed sites of physiological ligand-interaction, and therefore may disrupt critical ligand-receptor interactions in the host.
It is clear that it is not yet known which, if any, of the above antitoxins (or combinations thereof) will be effective at eliminating the risk posed by exposure to high systemic levels of anthrax toxin in the bloodstream. This is of particular concern when one considers the use of engineered organisms in a biological attack. Thus, it seems prudent to further develop additional antitoxins or a “cocktail” of antitoxins that, when used together, will be far more effective than would any one alone. It would also seem prudent to develop additional antitoxins capable of inhibiting anthrax toxin activity while not having a deleterious effect on the host.